1. Field of the Invention
The present invention relates to the detection of the presence of reverse transcriptase enzymatic activity in eukaryotic cell lines. Specifically, the present invention relates to a method for the discrimination of endogenous reverse transcriptase activity in avian cell lines used as substrates for the manufacture of biological products including viral vaccines from exogenous infectious retrovirus.
2. Summary of the Related Art
Of the commonly used viral vaccines licensed in the US, measles, mumps, yellow fever and influenza are produced in chicken cells derived from embryonated eggs (Plotkin and Mortimer (1994) Vaccines 2nd Edition, Philadelphia, Pa., Saunders). Furthermore, cell substrates can be used for production of a variety of biological products, such as vaccines for hepatitis A, polio, and rabies as well as proteins, glycoproteins, and lipoproteins. The use of living cell substrates for the production of live attenuated virus provides the opportunity for infection of these cell lines by adventitious retroviruses. These live attenuated vaccines may act as vehicles for the transmission of different kinds of microorganisms or other undesired agents. The risk for inadvertent transmission is particularly high for live attenuated virus vaccine since they cannot be subjected to an inactivation procedure and most of them are injected into human, thus by-passing non-specific immune protection mechanisms. Thus, to ensure safety of vaccines for human use the cell substrates for vaccine production have been tested for the presence of replication-competent retroviruses that could be passed to human hosts during immunization (WHO Technical Report Series, 1994). Vaccines produced in continuous cell lines of any origin are required by experts in the field to be free of RT activity (Boni et al., (1996) Clin. and Diag. Virol. 5: 43-53.)
Avian cell lines are frequently used in the production of vaccines. Avian retroviruses classified within the ALSV subgroup E have been shown to be maintained in the germ line as endogenous virus (ev) loci and passed to progeny in a Mendelian fashion [16]. Chickens bred to be resistant to the ALSV subgroup E endogenous viruses (e.g., line-0 chickens), and therefore lacking ALV (ev) loci, have been shown to carry other germ line sequences, some of which are believed to be remnants of ancient proviral genomes. Another family of endogenous avian retroviruses of interest is referred to as (EAV-0) since they were first isolated from the (evxe2x88x92) genome of line-0 chickens [17-20]. The EAV-0 loci have been shown to be highly expressed in embryonic cells derived from the avian genus, Gallus [21]. Moreover, these particles have been shown to contain RNA genomes with active RT-primer tRNA. Recent studies have shown that the particles from the EAV-0 endogenous retrovirus family are most likely responsible for a large portion of the particle-associated RT activity found in supernatants of cultured chick embryo fibroblasts [21]. Sequence analysis of several different EAV-0 isolates have indicated that a large deletion has occurred in the env gene which provides the scientific basis for and supports direct evidence for the replication incompetence of the particle-associated RT activity in cell culture [21].
Testing for the presence of retrovirus in cell substrates has been accomplished by assaying for the presence of reverse transcriptase (RT) activity based on the RNA-dependent synthesis of DNA (U.S. Pat. No. 3,755,086; Poiesz et al., (1980) Proc. Natl. Acad. Sci. USA, 77: 1415; Hoffman et al., (1985) Virology 147: 326). The RT enzyme or RNA-dependent DNA polymerase is essential for the establishment of the proviral state after infection of a cell and therefore is a mandatory component of the virus particle. Unfortunately, regardless of the templates and primers used, these RT assays are relatively insensitive compared to other more time-consuming assays for the detection of retroviruses such as cell culture or antigen detection methods. Attempts to detect uncharacterized retroviruses by the use of the polymerase chain reaction (PCR) with primers from conserved genomic regions have met with some success, but they lack the ability to assay for more than one infectious virus at a time, that is, the PCR test is not an open test. Furthermore, PCR will not detect the presence of unknown virus or viruses that have mutated in the region of the genome used for PCR amplification. PCR methods that utilize primers to specific retroviruses may not detect mutant variations of that virus if the mutation occurs in area to which the primers hybridize. Furthermore, PCR methods that utilize primers to specific retroviruses may require multiple and time consuming iterations to detect all possible retroviral species that may infect the substrate cell. Thus, both traditional RT assay and PCR based detection of retrovirus based on amplification of specific sequences are both relatively insensitive and can be time-consuming and difficult.
Recently, highly sensitive PCR based assays have been developed that can detect RNA-dependent DNA polymerase in the equivalent of one to ten particles (Silver et al. (1993) Nucleic Acids Res. 21: 3593-4; U.S. Pat. No. 5,807,669). One such assay, designated as PBRT (PCR-based reverse transcriptase), has been used to detect RT activity in a variety of samples (Pyra et al. (1994) Proc. Natl. Acad. Sci. USA 51: 1544-8; Boni, et al. (1996) J. Med. Virol. 49: 23-28). This assay is 106-107 more sensitive than the conventional RT assay. In one instance PBRT was used to detect RT activity in live attenuated viral vaccines grown in chicken cells (Boni et al. (1996) Clin. Diag. Virol. 5: 43-53). The presence of RT activity in avian substrate cells where none had previously been detected is a potential source of concern since this activity may indicate the presence of a retrovirus that can potentially replicate in human cells. Thus, administration of a licensed viral vaccine may be the conduit for the inadvertent administration an adventitious retrovirus that can infect human cells and persist long-term as an integral part of the host genome. This is accomplished through the retroviral replication cycle that is characteristic of all retroviruses and depends in part upon the enzymatic activity of RT (Varmus and Swanstrom (1984) RNA Tumor Viruses, Molecular Biology of Tumor Viruses vol 2, 2nd Edition, New York, Cold Spring Harbor Laboratory pp. 75-134; Baltimore (1970) Nature 226: 1209-11). Since retroviruses can be pathogenic in susceptible species, vaccines and other biological products for human use should be free of infectious retroviruses.
Alternatively, the presence of RT activity associated with cell substrates used in the production of viral vaccines may be due to low levels of RT-like activity associated with polymerases endogenous to the cell substrates. Moreover, it has recently been discovered that at least some of the RT activity associated with chicken cells used as substrates in the manufacture of live attenuated viral vaccines is associated with a particle that contains RNAs related to the EAV-0 family of retroviruses (Weissmahr et al. (1997) J. Virol. 71: 3005-12). Sequences of the EAV-0 family are prevalent in the genus Gallus and have been described as ancient proviral genomes (Dunwiddie et al. (1986) J. Virol. 59: 669-75); Resnick et al. (1990) J. Virol. 64: 4640-53; Boyce-Jacino et al. (1992) J. Virol. 66: 4919-29). Early studies using traditional RT assays had reported a particle-associated RT activity in the allantoic fluid of embryonated chicken eggs from retrovirus-free chickens and in concentrated medium of primary and secondary chicken embryo fibroblast cultures (Bauer and Hoffschneider (1977) Proc. Natl. Acad. Sci. USA 73: 3025-9; Bauer et al (1977) Eur. J. Biochem. 79: 345-54; Bauer et al. (1978) Biochim. Biophys. Acta 518: 125-37). No infectious retrovirus was found associated with the RT activity using avian target cells (Bauer et al. (1978) Exp. Cell Res. 117: 383-92).
Thus, utilizing the more sensitive PBRT assay testing of viral vaccine substrate cells for the presence of exogenous RT activity is problematic in that there is an increased incidence of false positive results due to the presence of endogenous RT activity. Several investigators have attempted to address the problem of false positives in the PBRT assay by modifying the assay conditions to decrease the RT activity produced by certain non-RT polymerases. Mandru and Peden (J. Virol. Methods, 66: 247-61 (1997)) were able to decrease the level of background RT activity produced by the RNA-dependent DNA polymerase activity of Taq polymerase by inserting a ribonucleotide digestion step prior to amplification of the cDNA product of the RT reaction by PCR and by using a thermostable DNA polymerase identified as having reduced RNA-dependent DNA polymerase activity. Chang et al. (J. Virol. Methods, 65: 45-54 (1997)) describe a PBRT assay in which the pH of the RT reaction was lowered, the incubation time of the RT reaction was decreased, and protease inhibitors were added to the RT reaction to decrease the RT activity of contaminating cellular polymerases. Lugert et al. (Biotechniques 20: 210-7 (1996)) describe a modified RT-PCR assay in which the amount of activated DNA was increased to inhibit the RT activity associated with endogenous polymerases but did not affect the RT activity of authentic RT molecules. None of the above referenced PBRT assays can distinguish RT activity from an adventitious retrovirus that may infect human cells from an endogenous RT source that is non-infectious, such as EAV-0.
Therefore, there exists a need for a screening method for RT activity in substrate cells that are used in the production of biological products, especially live attenuated virus for vaccines that can distinguish between non-infectious endogenous RT activity and RT activity associated with exogenous infectious retroviruses.
The present invention provides a method for detecting exogenous reverse transcriptase (RT) activity in a biological sample without interference from endogenous RT activity. This method is of particular use when the cellular substrate has a high level of endogenous RT activity (e.g., avian cells), making it impossible to assess the presence of contaminating infectious retroviruses based solely on the signature of RT activity. The present method achieves this by coupling an optimized transmissibility assay (for infectious retroviral amplification) with an optimized PBRT assay for detecting RT activity. The number of PCR cycles employed in the PBRT assay is selected to permit detection of the RT activity from the amplified exogenous retrovirus but not for detection of the lower level endogenous RT activity. The coupling of a retrovirus amplification process with PBRT is called Co-RT (CoRT) to reflect the coordination of the two assays.
Preliminarily, one determines the number of PCR cycles in the PBRT assay necessary to detect endogenous RT activity in a chosen cellular substrate permissive to retroviral infection, which substrate is to be used for exogenous infectious retrovirus amplification. Then, the transmissibility assay is employed to determine the amplification parameters required to yield an RT activity level greater than the endogenous RT activity of the cellular substrate and that can be detected with the PBRT assay using a number of PCR cycles that is less than the minimum required to detect endogenous RT activity of the cellular substrate. The degree of amplification necessary as determined by the transmissibility assay will, of course, depend both on the endogenous RT activity as well as on the initial level of exogenous infectious RT activityxe2x80x94the lower the level of exogenous infectious RT activity relative to the level of endogenous RT activity, the greater the amount of amplification necessary. For any particular amplification protocol, there will be a threshold level of detectable exogenous RT activity in the CoRT assay. The threshold level of exogenous infectious retrovirus detectable with a given set of CoRT assay parameters is that amount which after amplification yields an RT activity detectable by a number of PCR cycles in the PBRT assay that is at least one less than that required to detect endogenous RT activity.
Once the parameters for amplification from the transmissibility assay and detection from the PBRT assay have been determined, the CoRT assay according to the invention can be routinely and repeatedly conducted on biological samples of interest using those parameters. As demonstrated herein, the sensitivity achieved with 1.0 TCID50 using the CoRT assay is similar to that achieved using the specific PCR assays.
The foregoing merely summarizes certain aspects of the invention and is not intended to limit the invention. All patents and other publications are hereby incorporated by reference in their entirety.